Cyclooxygenase (COX) inhibitors lower the risk of colorectal cancer (CRC) and inhibit tumor growth in animal and cell culture models. However, their efficacy in treating existing CRC remains controversial. Previous research in our laboratory has demonstrated that stromal colonic myofibroblasts (CMFs), and not epithelial cells are the principal COX-2 expressing cells in human colonic adenomas and adenocarcinomas. Wnt/&#946;catenin signaling is a crucial pathway for the development of CRC and over 90% of sporadic colorectal adenocarcinomas harbor activating mutations along this axis. CMFs also synthesize paracrine factors capable of augmenting or suppressing epithelial Wnt signaling. Wnt signaling is also crucial for maintenance of intestinal epithelial stem cells and we have shown that CMFs contribute to the epithelial stem cell niche of the normal colon. We have isolated CMFs from normal human colonic tissue and colorectal adenocarcinomas and have identified distinct phenotypic differences between them. In the research proposed, we will test the hypothesis that in normal colonic mucosa and colorectal cancer, alterations in paracrine Wnt/&#946;catenin and bone morphogenetic protein (BMP) family signaling between the colonic epithelium and myofibroblasts create a microenvironment that favors neoplasia. These alterations affect not only proliferation of the cancerous epithelium, but also tumor initiating cell population dynamics. We will test our hypothesis by achieving the following Specific Aims: Aim 1. Determine how CMFs derived from normal or neoplastic tissues differ in their ability to modulate epithelial Wnt/&#946;catenin signaling directly via production of Wnt pathway agonists and antagonists. Aim 2. Determine how CMFs derived from normal or neoplastic tissues differ in their ability to modulate epithelial Wnt/&#946;catenin signaling indirectly via production of BMP pathway agonists and antagonists. Aim 3. Identify how CMF-derived Wnt and BMP modulators affect tumor initiating cell (cancer stem cell) proliferation and survival. The long term goals of this project are to develop more effective therapeutic regimens by targeting not only malignant epithelial cells, but also the tumor initiating or cancer stem cells (CSC) responsible for tumor recurrence and the stromal cells (myofibroblasts) that nurture tumor growth and progression as well as the cancer stem cell niche.